Through the eye of an electrospray needle: mass spectrometric identification of the major peptides and proteins in the milk of the one‐humped camel (Camelus dromedarius)

The milk of the one‐humped camel (Camelus dromedarius) reportedly offers medicinal benefits, perhaps because of its unique bioactive components. Milk proteins were determined by (1) two‐dimensional gel electrophoresis and peptide mass mapping and (2) liquid chromatography–tandem mass spectrometry (LC–MS/MS) following one‐dimensional polyacrylamide gel electrophoresis. Over 200 proteins were identified: some known camel proteins including heavy‐chain immunoglobulins and others exhibiting regions of exact homology with proteins from other species. Indigenous peptides were also identified following isolation and concentration by two strategies: (1) gel‐eluted liquid fraction entrapment electrophoresis and (2) small‐scale electrophoretic separation. Extracts were analyzed by LC–MS/MS and peptides identified by matching strategies, by de novo sequencing and by applying a sequence tag tool requiring similarity to the proposed sequence, but not an exact match. A plethora of protein cleavage products including some novel peptides were characterized. These studies demonstrate that camel milk is a rich source of peptides, some of which may serve as nutraceuticals. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterisation of low abundance wool proteins through novel differential extraction techniques

Fibres from human hair and wool are characterised by two main types of proteins: intermediate filament proteins (IFPs) and keratin associated proteins (KAPs). The IFPs, comprising over 50% of the fibre, tend to dominate 2‐D electrophoretic maps, hindering identification of the less‐abundant KAPs. This has been compounded in wool fibres by the relatively limited amount of sequence information available, with approximately 35 distinct protein sequences from ten KAP families being available, in contrast to human hair, where the sequences from well over 80 proteins from 26 KAP families are known. Additional complications include the high degree of homology within these families, ranging from 70 to 95%, and the dominance of cysteine residues in a number of KAP families with their high propensity to form cross‐links. The lack of sequence information for wool KAPs has been partly overcome through the recent acquisition of new sequences. Fractionation of the proteins on the basis of their solubility with pH, urea and DTT concentration has resulted in protein extracts in which the IFP concentration has been considerably reduced. These improvements have enabled the identification of low‐abundance proteins in 2‐D electrophoretic maps and represent a significant advance in our knowledge of the wool proteome.     

Multidimensional persistence in biomolecular data

Persistent homology has emerged as a popular technique for the topological simplification of big data, including biomolecular data. Multidimensional persistence bears considerable promise to bridge the gap between geometry and topology. However, its practical and robust construction has been a challenge. We introduce two families of multidimensional persistence, namely pseudomultidimensional persistence and multiscale multidimensional persistence. The former is generated via the repeated applications of persistent homology filtration to high‐dimensional data, such as results from molecular dynamics or partial differential equations. The latter is constructed via isotropic and anisotropic scales that create new simiplicial complexes and associated topological spaces. The utility, robustness, and efficiency of the proposed topological methods are demonstrated via protein folding, protein flexibility analysis, the topological denoising of cryoelectron microscopy data, and the scale dependence of nanoparticles. Topological transition between partial folded and unfolded proteins has been observed in multidimensional persistence. The separation between noise topological signatures and molecular topological fingerprints is achieved by the Laplace–Beltrami flow. The multiscale multidimensional persistent homology reveals relative local features in Betti‐0 invariants and the relatively global characteristics of Betti‐1 and Betti‐2 invariants. © 2015 Wiley Periodicals, Inc.     

Protein Delivery System Containing a Nickel‐Immobilized Polymer for Multimerization of Affinity‐Purified His‐Tagged Proteins Enhances Cytosolic Transfer

Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea‐grafted polyethylenimine (πPEI) with affinity‐purified His‐tagged proteins pre‐organized onto a nickel‐immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His‐tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His‐tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single‐chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions.     

LEAP: Highly accurate prediction of protein loop conformations by integrating coarse‐grained sampling and optimized energy scores with all‐atom refinement of backbone and side chains

Prediction of protein loop conformations without any prior knowledge (ab initio prediction) is an unsolved problem. Its solution will significantly impact protein homology and template‐based modeling as well as ab initio protein‐structure prediction. Here, we developed a coarse‐grained, optimized scoring function for initial sampling and ranking of loop decoys. The resulting decoys are then further optimized in backbone and side‐chain conformations and ranked by all‐atom energy scoring functions. The final integrated technique called loop prediction by energy‐assisted protocol achieved a median value of 2.1 Å root mean square deviation (RMSD) for 325 12‐residue test loops and 2.0 Å RMSD for 45 12‐residue loops from critical assessment of structure‐prediction techniques (CASP) 10 target proteins with native core structures (backbone and side chains). If all side‐chain conformations in protein cores were predicted in the absence of the target loop, loop‐prediction accuracy only reduces slightly (0.2 Å difference in RMSD for 12‐residue loops in the CASP target proteins). The accuracy obtained is about 1 Å RMSD or more improvement over other methods we tested. The executable file for a Linux system is freely available for academic users at http://sparks‐lab.org . © 2013 Wiley Periodicals, Inc.     

Analysis of variation in the ovine ultra‐high sulphur keratin‐associated protein KAP5‐4 gene using PCR‐SSCP technique

Keratin‐associated proteins (KAPs) are one of the main structural components of the wool fibre. Variation in the KAP genes (KRTAPs) may affect the structure of KAPs and hence wool characteristics. In this study, we used PCR‐SSCP to analyse ovine KRTAP5‐4, a gene encoding a member of the KAP5 family. Five different PCR‐SSCP patterns were detected in the 250 sheep that were analysed. Either one or a combination of two patterns was observed for each sheep, which was consistent with these sheep being either homozygous or heterozygous at this locus. DNA sequencing revealed that these patterns represent five different DNA sequences. One of the sequences was identical to a published ovine KRTAP5‐4 sequence. The remaining four were unique, but shared a high homology with the published ovine KRTAP5‐4 sequence, suggesting that these sequences represent allelic variants of KRTAP5‐4. There were a total of six SNPs and one length polymorphism in the sequences. Of the five SNPs found in the coding region, four were non‐synonymous SNPs and would result in amino acid changes. The length polymorphism would affect the cysteine content of the putative peptide and this along with the SNPs may have an impact on the structure of KAP5‐4, and hence affect wool traits.     

IR Probes of Protein Microenvironments: Utility and Potential for Perturbation

A variety of IR‐active moieties with absorptions that are distinct from those of proteins have been developed as probes of local protein environments, including carbon‐deuterium bonds (C?D), cyano groups (CN), and azides (N₃); however, no systematic analysis of their utility in a protein has been published. Previously, we characterized the N‐terminal Src homology 3 domain of the murine adapter protein Crk‐II (nSH3) with C?D bonds site‐selectively incorporated throughout, and showed that it is relatively rigid and electrostatically heterogeneous and that it thermally unfolds under equilibrium conditions via a simple two‐state mechanism. We now report the synthesis and characterization of eight variants of nSH3 with CN and/or N₃ probes at five of the same positions. In agreement with previous studies, the position‐dependent spectra suggest that both probes are predominantly sensitive to hydration, and not to their local electrostatic environments. Importantly, both probes also tend to significantly perturb the protein if they are not incorporated at surface‐exposed positions. Thus, unlike C?D labels, which are both sensitive to their environment and non‐perturbative, CN and N₃ probes should be used with caution.     

Intracellular Delivery of Native Proteins Facilitated by Cell‐Penetrating Poly(disulfide)s

Intracellular delivery of therapeutic proteins is highly challenging and in most cases requires chemical or genetic modifications. Herein, two complementary approaches for endocytosis‐independent delivery of proteins to live mammalian cells are reported. By using either a “glycan” tag naturally derived from glycosylated proteins or a “traceless” tag that could reversibly label native lysines on non‐glycosylated proteins, followed by bioorthogonal conjugation with cell‐penetrating poly(disulfide)s (CPDs), we achieved intracellular delivery of proteins (including antibodies and enzymes) which, upon spontaneous degradation of CPDs, led to successful release of their “native” functional forms with immediate bioavailability.     

Alpha‐1 antitrypsin variants in plasma from HIV‐infected patients revealed by proteomic and glycoproteomic analysis

Novel tools are necessary to explore proteins related to human immunodeficiency virus (HIV) infection. In this work, proteomic and glycoproteomic technology were employed to examine plasma samples from HIV‐positive patients. Through comparative proteome analysis of normal and HIV‐positive plasma samples, 19 differentially expressed protein spots related to 12 non‐redundant proteins were identified by ESI‐ion trap MS. Among these, the 130‐kDa isoform of α‐1‐antitrypsin was found to be decreased in HIV‐positive patients while another variant with a molecular weight of 40 kDa was increased. SWISS‐2‐D‐PAGE reference gel and protein sequence comparisons of the 40‐kDa protein showed homology with α‐1‐antitrypsin minus the N‐terminus, and its identity was further confirmed by 1‐D Western blotting and glycoproteomic analysis. In all, our results showed that proteomics and glycoproteomics are powerful tools for discovering proteins related to HIV infection. Furthermore, this 40‐kDa variant of α‐1‐antitrypsin found in the plasma of HIV‐positive individuals may prove to be a potentially useful biomarker for anti‐HIV research according to bioinformatics analysis.     

RNA-binding residues in sequence space: Conservation and interaction patterns

RNA-binding proteins (RBPs) perform fundamental and diverse functions within the cell. Approximately 15% of proteins sequences are annotated as RNA-binding, but with a significant number of proteins without functional annotation, many RBPs are yet to be identified. A percentage of uncharacterised proteins can be annotated by transferring functional information from proteins sharing significant sequence homology. However, genomes contain a significant number of orphan open reading frames (ORFs) that do not share significant sequence similarity to other ORFs, but correspond to functional proteins. Hence methods for protein function annotation that go beyond sequence homology are essential. One method of annotation is the identification of ligands that bind to proteins, through the characterisation of binding site residues. In the current work RNA-binding residues (RBRs) are characterised in terms of their evolutionary conservation and the patterns they form in sequence space. The potential for such characteristics to be used to identify RBPs from sequence is then evaluated.In the current work the conservation of residues in 261 RBPs is compared for (a) RBRs vs. non-RBRs surface residues, and for (b) specific and non-specific RBRs. The analysis shows that RBRs are more conserved than other surface residues, and RBRs hydrogen-bonded to the RNA backbone are more conserved than those making hydrogen bonds to RNA bases. This observed conservation of RBRs was then used to inform the construction of RBR sequence patterns from known protein–RNA structures. A series of RBR patterns were generated for a case study protein aspartyl-tRNA synthetase bound to tRNA; and used to differentiate between RNA-binding and non-RNA-binding protein sequences. Six sequence patterns performed with high precision values of >80% and recall values 7 times that of an homology search. When the method was expanded to the complete dataset of 261 proteins, many patterns were of poor predictive value, as they had not been manipulated on a family-specific basis. However, two patterns with precision values ≥85% were used to make function predictions for a set of hypothetical proteins. This revealed a number of potential RBPs that require experimental verification.     

SPOT‐Ligand: Fast and effective structure‐based virtual screening by binding homology search according to ligand and receptor similarity

Structure‐based virtual screening usually involves docking of a library of chemical compounds onto the functional pocket of the target receptor so as to discover novel classes of ligands. However, the overall success rate remains low and screening a large library is computationally intensive. An alternative to this “ab initio” approach is virtual screening by binding homology search. In this approach, potential ligands are predicted based on similar interaction pairs (similarity in receptors and ligands). SPOT‐Ligand is an approach that integrates ligand similarity by Tanimoto coefficient and receptor similarity by protein structure alignment program SPalign. The method was found to yield a consistent performance in DUD and DUD‐E docking benchmarks even if model structures were employed. It improves over docking methods (DOCK6 and AUTODOCK Vina) and has a performance comparable to or better than other binding‐homology methods (FINDsite and PoLi) with higher computational efficiency. The server is available at http://sparks-lab.org . © 2016 Wiley Periodicals, Inc.     

A Double‐Armed,Hydrophilic Transition Metal Complex as a Paramagnetic NMR Probe

Synthetic metal complexes can be used as paramagnetic probes for the study of proteins and protein complexes. Herein, two transition metal NMR probes (TraNPs) are reported. TraNPs are attached through two arms to a protein to generate a pseudocontact shift (PCS) using cobalt(II), or paramagnetic relaxation enhancement (PRE) with manganese(II). The PCS analysis of TraNPs attached to three different proteins shows that the size of the anisotropic component of the magnetic susceptibility depends on the probe surroundings at the surface of the protein, contrary to what is observed for lanthanoid‐based probes. The observed PCS are relatively small, making cobalt‐based probes suitable for localized studies, such as of an active site. The obtained PREs are stronger than those obtained with nitroxide spin labels and the possibility to generate both PCS and PRE offers advantages. The properties of TraNPs in comparison with other cobalt‐based probes are discussed.     

p‐Azidophenylarsenoxide: An Arsenical “Bait” for the In Situ Capture and Identification of Cellular Arsenic‐Binding Proteins

Identification of arsenic‐binding proteins is important for understanding arsenic health effects and for developing arsenic‐based therapeutics. We report here a strategy for the capture and identification of arsenic‐binding proteins in living cells. We designed an azide‐labeled arsenical, p‐azidophenylarsenoxide (PAzPAO), to serve bio‐orthogonal functions: the trivalent arsenical group binds to cellular proteins in situ, and the azide group facilitates click chemistry with dibenzylcyclooctyne. The selective and efficient capture of arsenic‐binding proteins enables subsequent enrichment and identification by shotgun proteomics. Applications of the technique are demonstrated using the A549 human lung carcinoma cells and two in vitro model systems. The technique enables the capture and identification of 48 arsenic‐binding proteins in A549 cells incubated with PAzPAO. Among the identified proteins are a series of antioxidant proteins (e.g., thioredoxin, peroxiredoxin, peroxide reductase, glutathione reductase, and protein disulfide isomerase) and glyceraldehyde‐3‐phosphate dehydrogenase. Identification of these functional proteins, along with studies of arsenic binding and enzymatic inhibition, points to these proteins as potential molecular targets that play important roles in arsenic‐induced health effects and in cancer treatment.     

Electrophoretic mapping of highly homologous keratins: A novel marker peptide approach

Identification of the intermediate filament proteins (IFPs) in the wool proteome has formerly been hampered by limited sequence information, the high degree of IFP homology and their close proximity on 2‐DE maps. This has been partially rectified by the recent acquisition of four new Type I and two Type II wool IFP sequences. Among closely migrating proteins, such as IFP clusters in a 2‐DE map, proteins with higher sequence coverage will be assigned higher scores, but the identification of unique peptides in such tight clusters may distinguish these closely migrating proteins. Two approaches were adopted for the study of wool IFPs. In the first, searches were conducted for peptides known to be unique to each member of the family in each spot. In the second, MALDI imaging was employed to examine peptides bound to a PVDF membrane from a poorly resolved part of the Type I IFP region of the 2‐DE map. As a result, a distinct picture has emerged of the distribution of the six Type I and four Type II IFPs across the 2‐DE wool protein map.     

Comparison of the structures of human fibronectin and plasma cold-insoluble globulin

Human amniotic fluid fibronectin and plasma fibronectin (cold-insoluble globulin) are indistinguishable both immunologically and by amino acid composition. Cyanogen bromide and tryptic peptides also suggest substantial structural homology. However, carbohydrate analysis has demonstrated additional saccharides in fibronectin and an overall increase in carbohydrate content relative to cold-insoluble globulin. Furthermore, limited proteolytic cleavage of the two proteins indicates differences in primary structure or in conformation. Using affinity-purified antibodies to cold-insoluble globulin, a glucosamine-labeled pronase-resistant component, probably proteoglycan, was found to coprecipitate with fibronectin, suggesting an association between these two macromolecules in the connective tissue matrix.     

A Double‐Armed,Hydrophilic Transition Metal Complex as a Paramagnetic NMR Probe

Synthetic metal complexes can be used as paramagnetic probes for the study of proteins and protein complexes. Herein, two transition metal NMR probes (TraNPs) are reported. TraNPs are attached through two arms to a protein to generate a pseudocontact shift (PCS) using cobalt(II), or paramagnetic relaxation enhancement (PRE) with manganese(II). The PCS analysis of TraNPs attached to three different proteins shows that the size of the anisotropic component of the magnetic susceptibility depends on the probe surroundings at the surface of the protein, contrary to what is observed for lanthanoid‐based probes. The observed PCS are relatively small, making cobalt‐based probes suitable for localized studies, such as of an active site. The obtained PREs are stronger than those obtained with nitroxide spin labels and the possibility to generate both PCS and PRE offers advantages. The properties of TraNPs in comparison with other cobalt‐based probes are discussed.     

Extracellular SH3 domain containing proteins--features of a new protein family

In the year 1994, the protein MIA (melanoma inhibitory activity) was found to be strongly expressed and secreted by malignant melanomas and subsequent studies revealed that MIA has an important function in melanoma development and invasion. Multidimensional NMR-spectroscopy and x-ray crystallography revealed that recombinant human MIA adopts a Src homology 3 (SH3) domain-like fold in solution, a structure with two perpendicular antiparallel three- and five-stranded beta-sheets. SH3 domains are protein modules that are found in many intracellular signalling proteins and mediate protein-protein interactions by binding to proline-rich peptide sequences. Unlike previously described protein structures with SH3 domain folds, MIA is a secreted single-domain protein of 12 kDa that contains an additional antiparallel beta-sheet and two disulfide bonds. Furthermore, the charge surrounding the canonical binding site differs from that of classical SH3 domains. The two disulfide bonds are crucial for correct folding and function as revealed by mutation analysis. Therefore, MIA appears to be the first extracellular protein adopting an SH3 domain-like fold. MIA was shown to interact with fibronectin, and MIA-interacting peptide ligands identified by phage display screening are similar to the consensus sequence of type III human fibronectin repeats, especially FN14. Interestingly, recent data revealed that MIA can also directly bind to integrin alpha 4 beta 1 and alpha 5 beta1 and that it modulates integrin activity negatively. These findings suggest an interesting role of the SH3-domain proteins in the extracellular compartment. Recently, MIA homologous proteins with a sequence identity of 44% and a sequence homology of approximately 80% were determined (TANGO, MIA-2, OTOR). This clearly suggests that this structural device is used more frequently, in processes ranging from developmental changes to the interference of cell attachment in the extracellular matrix. Detailed studies are necessary to determine the exact function of the MIA homologous proteins. It will be interesting to know whether additional protein families can be identified which are secreted and carry SH3 domain-like modules, in addition to elucidate what the specific physiological targets of this protein family are.